Herpes simplex virus type 2 and cytomegalovirus perigenital ulcer in an HIV infected woman

We report a case of mucocutaneous Herpes Simplex Virus (HSV)-2 and Cytomegalovirus (CMV) infection in a 39-year-old female with acquired immunodeficiency syndrome, who presented with a perigenital ulcer. The patient was receiving antiretroviral treatment (ART) for 3 months before presentation. Scraping from the perigenital ulcer was positive for HSV-2 and Treponema pallidum using polymerase chain reactions (PCR). The extent and duration of the lesions led us to consider the possibility of coinfection with CMV. The patient also tested positive for CMV by PCR. On subsequent follow-up after 8 weeks, the genital lesions had healed completely. This is possibly ascribable to the ART, which led to significant immune reconstitution.

Comparison of conventional, immunological and molecular techniques for the diagnosis of symptomatic congenital human cytomegalovirus infection in neonates and infants.

Purpose: Human cytomegalovirus (HCMV) is the commonest pathogen causing congenital infection globally. The diagnosis of congenital infection is based either on viral isolation (in cell culture) or demonstration of HCMV DNA from the urine. Saliva is also being used as an alternative sample to urine for the same. The objective of this study was to compare the following assays-polymerase chain reaction (PCR) from urine, saliva and blood, serology (anti-HCMV IgM) and antigen detection (HCMV pp65 antigenaemia) for the diagnosis of congenital HCMV infection. Materials and Methods: Urine and blood samples were collected from 31 infants (median age: 13 weeks) with suspected HCMV infection. For 18 infants, additional saliva samples were collected and all the above assays were compared. Results: PCR for HCMV DNA from urine and anti-HCMV IgM were performed for all 31 infants. Of these, 22 (70.9%) were positive for both assays. In 18 (of the 22) infants positive by both assays, PCR for HCMV DNA from saliva was positive in all 18 (100%), PCR from blood in 7/18 (38.8%) and HCMV pp65 antigenaemia only in 1/18 (5.5%) of the infants. Conclusion: Detection of HCMV DNA in urine combined with anti-HCMV IgM are suitable assays to diagnose HCMV infection in infants. Both PCR from the blood and HCMV pp65 antigenaemia lack sensitivity in infants. Salivary PCR combines convenience with high sensitivity and can substitute PCR from urine, especially in the outpatient and fi eld settings. To the best of our knowledge, this is the fi rst study from India to evaluate salivary PCR for the diagnosis of congenital HCMV infection.

Dengue vaccines: Challenges, development, current status and prospects.

Infection with dengue virus (DENV) is the most rapidly spreading mosquito-borne viral disease in the world. The clinical spectrum of dengue, caused by any of the four serotypes of DENV, ranges from mild self-limiting dengue fever to severe dengue, in the form dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased rates of hospitalization due to severe dengue, during outbreaks, result in massive economic losses and strained health services. In the absence of specifi c antiviral therapy, control of transmission of DENV by vector management is the sole method available for decreasing dengue-associated morbidity. Since vector control strategies alone have not been able to satisfactorily achieve reduction in viral transmission, the implementation of a safe, effi cacious and cost-effective dengue vaccine as a supplementary measure is a high public health priority. However, the unique and complex immunopathology of dengue has complicated vaccine development. Dengue vaccines have also been challenged by critical issues like lack of animal models for the disease and absence of suitable markers of protective immunity. Although no licensed dengue vaccine is yet available, several vaccine candidates are under phases of development, including live attenuated virus vaccines, live chimeric virus vaccines, inactivated virus vaccines, subunit vaccines, DNA vaccines and viral-vectored vaccines. Although some vaccine candidates have progressed from animal trials to phase II and III in humans, a number of issues regarding implementation of dengue vaccine in countries like India still need to be addressed. Despite the current limitations, collaborative effects of regulatory bodies like World Health Organization with vaccine manufacturers and policy makers, to facilitate vaccine development and standardize fi eld trials can make a safe and effi cacious dengue vaccine a reality in near future.

Quality assurance of annual HIV sentinel surveillance 2006: experience of regional institute, central zone.

National AIDS Control Organisation (NACO) identified five regional institutes (RIs) to monitor and supervise the 2006 round of annual HIV sentinel surveillance. The task mandated was quality control of both epidemiological data collection and HIV testing. The team at RI consisted of epidemiologist and microbiologist. We describe here the process of quality control and the quality of surveillance in the states of Uttar Pradesh, Uttarakhand, Bihar, Jharkand, and Delhi. The supervisors visited almost 90% of the sentinel sites. Performance of vast majority of the sentinel sites (92%) was satisfactory. The testing laboratories were found to be adhering to standard operating procedures. Concordance rate of test results between testing laboratory and the designated reference laboratory was high. Overall, the quality of sentinel surveillance was good. The lacunae found during the visit have been enumerated along with the recommendations for future surveillance round.

Role of CSF serology in follow-up of subacute sclerosing panencephalitis patients on treatment.

Subacute sclerosing panencephalitis (SSPE) is a progressive inflammatory disease of the central nervous system with poor prognosis and high mortality. No effective treatment has a proven role; oral isoprinosine and intrathecal administration of alpha-interferon may prolong survival. We report an unusual case of adult onset SSPE patient on treatment with significant clinical improvement, even in the absence of conversion to seronegativity in either CSF or serum, on follow-up serological examination.

Expression and humoral immune response to hepatitis C virus using a plasmid DNA construct.

PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA) and immunofluorescence (IFA). The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

Levels of CD4 and CD8 among the inhabitants of Manipur, India.

Data on mean reference CD4 and CD8% is in general lacking in India. Manipur in the North-East India has high prevalence of HIV infection among the injecting drug users. This study was carried out to establish mean reference CD4 and CD8 cell count in normal and HIV infected individuals in our population, for use in interpretation of these prognostic markers in HIV infected persons. Whole blood sample was collected in EDTA from 14 normal and 23 HIV infected individuals. Fluorescence staining was carried out with FITC conjugated anti-CD4 and CD8 antibodies (Becton Dickinson) directly on whole blood, followed by single step lysis using commercial lysing solution (Optilyse C, Immunotec). The samples were analyzed by two-colour flow cytometry on Coulter Elite cytometer. It was observed that the mean CD4 and CD8 positive cells in normal healthy individuals were 36% (absolute 848/cumm) and 21% (absolute 427/cumm) respectively. The mean CD4% was significantly decreased in HIV infected individuals with a mean value of 13.4% (absolute 246/cumm), while the mean CD8% was significantly increased to 39.2% (absolute 660/cumm) in HIV infected individuals. A lower CD4+ cell count was also observed as compared to the western population among the normal healthy individuals. The mean CD4 and CD8 positive cells in normal healthy adult population were found to be 36% and 21% respectively, and 13.4% and 39.2% in HIV infected individuals respectively. These values should be considered when interpreting CD4 and CD8 counts in HIV infected individuals in this part of the country.

Dengue virus infection during post-epidemic period in Delhi, India.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are major public health problems in India. During the period following an epidemic, a study was carried out using virological and serological tests for confirmation of suspected cases of dengue virus infection in fever cases presenting to the All India Institute of Medical Sciences. Serum samples of suspected DF/DHF cases were processed from January to December 1997. In 37 samples from patients with fever of less than 5-day duration, received on ice, virus isolation was attempted in C6/36 clone of Aedes albopictus cell line, followed by indirect fluorescent antibody staining with monoclonal antibodies to dengue viruses 1 to 4. One hundred and forty-three serum samples from patients with more than 5 days fever were tested for dengue specific IgM antibody by either MAC-ELISA or a rapid immunochromatographic assay. Dengue virus type 1 was demonstrated by culture in 8 (21.6%) of 37 serum samples and IgM antibody could be detected in 42 (29.4%) of the 143 serum samples by the serological methods. The peak of dengue virus infection was seen from September to November 1997.

Myocardial dysfunction in children with dengue haemorrhagic fever.

BACKGROUND: Dengue fever/dengue haemorrhagic fever/dengue shock syndrome is a serious health problem in tropical countries. Intravascular fluid depletion due to capillary leak is presumed to be the cause of hypotension in dengue haemorrhagic fever. The treatment guidelines of the World Health Organization lay stress primarily on monitoring and fluid replacement therapy. During the 1996 epidemic in New Delhi, we observed problems in fluid management of such children and prospectively looked for myocardial dysfunction as an additional factor for hypotension. METHODS: Fifty-four children (< 12 years old) admitted to the All India Institute of Medical Sciences, New Delhi after 15 October 1996 with various grades of the disease, who were fit to be shifted to the echocardiography laboratory, were examined clinically and subjected to a detailed M-mode, 2-dimensional and colour doppler echocardiography. Ejection fractions (Teichholz/Modified Simpson's) and shortening fractions were calculated. RESULTS: Ejection fraction by modified Simpson's rule was reduced (< 50%) in 9/54 (16.7%) children; 2 of these had significant reductions (< 35%). These 9 children belonged to all stages of clinical severity. Three of these 9 children who had a repeat echocardiogram within 2 months of the illness had improved ejection fractions. CONCLUSION: The role of myocardial dysfunction remains to be defined as there was no correlation with clinical severity. Myocardial functions need to be assessed in patients with this disease, especially those who have persistent hypotension in spite of adequate hydration.

Diagnosis of pulmonary tuberculosis by polymerase chain reaction for MPB64 gene: an evaluation in a blind study.

Polymerase chain reaction (PCR) has been found to be a sensitive and rapid method to confirm a clinical diagnosis of tuberculosis. We evaluated PCR for M. tuberculosis complex specific MPB64 gene for the diagnosis of pulmonary tuberculosis, in a double blind study. One hundred and eighty-two clinical samples (sputum, bronchioalveolar lavage and pleural fluid) from patients with a clinical diagnosis of pulmonary tuberculosis and 72 samples from patients with non-tubercular pulmonary lesions and normal healthy individuals were included. The samples were coded and clinical details were concealed from the laboratory, where conventional diagnostic methods and PCR were carried out independent of each other. On decoding and analysing the data, PCR was positive in 59% of single sputum samples from clinically diagnosed pulmonary tuberculosis, while M. tuberculosis could be grown in 18% of the samples. PCR could identify M. tuberculosis in 81.8% of the culture positive sputum samples. PCR was also positive in 71.4% of bronchioalveolar lavage (BAL) fluid and 60.7% pleural fluid samples from clinically suspected cases, which were mostly culture negative. On comparison with response to treatment, PCR was positive in 79.5% of patients who improved on anti-tuberculosis treatment, with a positive predictive value of 92%. PCR for MPB64 gene provides a useful alternative for the diagnosis of pulmonary tuberculosis from sputum and paucibacillary samples like BAL and pleural fluid in which conventional methods show low sensitivity, especially in areas from which strains show a low copy number of other PCR targets like the IS 6110 insertion sequence.

Multidrug resistant Salmonella typhi in Delhi.

In 1990, we isolated 158 strains of Salmonella typhi from blood cultures of patients suffering from typhoid fever. Seventy nine (50%) of these isolates were found to be simultaneously resistant to chloramphenicol, ampicillin and cotrimoxazole. These strains were also resistant to streptomycin and tetracycline, but sensitive to gentamicin, amikacin and cephalexin. The minimum inhibitory concentrations of chloramphenicol and trimethoprim for a representative number of these strains were found to be greater than 1024 micrograms/ml and greater than 128 micrograms/ml respectively. Majority of the multidrug resistant (MDR) strains tested against cefotaxime (23/23), ciprofloxacin (38/38) and amoxycillin plus clavulanic acid (23/24) were sensitive to these drugs.